Materials and methods for detection and quantitation of an analyte

ABSTRACT

The subject invention pertains to methods and materials for accurately assessing the presence or absence of analytes of interest in samples, particularly in physiological samples. The subject invention involves utilizing a ligand binding domain (LBD) of a receptor to selectively capture the analyte target specific for that LBD. In one embodiment, the receptor is a protein or polypeptide. The ligand binding domain is allowed to react with a sample and the presence or amount of ligand (i.e., target analyte) bound by the LBD is determined. Suitable analytes include soluble analytes such as hormones, enzymes, lipoproteins, bacterial or viral antigens, immunoglobulines, lymphokines, cytokines, drugs, soluble cancer antigens, and the like. The methods of the present invention can be performed in both liquid-phase and solid-phase.

CROSS-REFERENCE TO A RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/660,979, filed Sep. 13, 2000, now U.S. Pat. No. 6,500,629, whichclaims the benefit of U.S. Provisional Application No. 60/153,627, filedSep. 13, 1999.

BACKGROUND OF THE INVENTION

There are a number of assay systems available for detection andquantitation of analytes, particularly analytes of biological interest.Current assay systems include enzyme immunoassay (EIA), radioimmunoassay(RIA), and enzyme linked immunosorbent assay (ELISA). Among the analytesfrequently assayed with such systems are: 1) hormones, such as humanchorionic gonadotropin (hCG), frequently assayed in urine as a marker ofhuman pregnancy; 2) antigens, particularly antigens specific tobacterial, viral, and protozoan pathogens, such as Streptococcus andhepatitis virus; 3) antibodies, particularly antibodies induced as aresult of infection with pathogens, such as antibody to the bacteriumHeliobacter pylori and to human immunodeficiency virus (HIV); 4)enzymes, such as aspartite aminotransferase, lactate dehydrogenase,alkaline phosphatase, and glutamate dehydrogenase, frequently assayed asindicators of physiological function and tissue damage; 5) otherproteins, such as hemoglobin, frequently assayed in determinations offecal occult blood, an early indicator of gastrointestinal disorderssuch as colon cancer; 6) drugs, both therapeutic drugs, such asantibiotics, tranquilizers and anticonvulsants, and illegal drugs ofabuse, such as cocaine, heroine, and marijuana; and 7) environmentalpollutants such as pesticides and aromatic hydrocarbons and vitamins.

Such systems are frequently used by physicians and medical techniciansfor rapid in-office diagnosis and therapeutic monitoring of a variety ofconditions and disorders. They are also increasingly used by patientsthemselves for at-home or on-site monitoring of such conditions anddisorders.

Among the most popular of such assay systems are immunoassays, whichdepend on the specific interaction between an antigen or hapten and acorresponding antibody. The use of immunoassays as a means of testingfor the presence and/or amount of clinically important molecules hasbeen known for some time. As early as 1956, J. M. Singer reported theuse of an immune-based latex agglutination test for detecting a factorassociated with rheumatoid arthritis (Singer et al., 1956).

Development of the first radioimmunoassay by Rosalyn Yalow and SolBerson (1959) set the stage for measurement of a wide variety ofhormones in biological fluids by binding the hormone specifically andwith high affinity to antibodies developed in animals against thehormone in question. The assay developed by Drs. Yalow and Bersonemployed antibodies formed against the protein hormone, insulin, andutilized a radiolabeled form of insulin as the marker, or “reporter”hormone. Antibodies became a useful way to “capture” a specific hormonefrom biological fluids and under conditions of constant antibodyconcentration and with some easily detected source of labeled hormone(usually radioactively labeled; hence the name “radioimmunoassay”) theamount of hormone “captured” from the biological fluid could bequantified by comparison to known concentrations of the hormone insimilar conditions. In practice of the art, known amounts of (unlabeled)hormone, (insulin in the example) were allowed to compete for binding tothe antibody with a known and fixed amount of I¹³¹ labeled insulin. Theradiolabeled form of hormone, and the amount of antibody were heldconstant while the amount of unlabeled hormone was varied. This was thebasis of a “standard curve” from which the amount of radioactive labelthat bound to the antibody varied inversely with the amount of unlabeledhormone. Comparison of the mass of unlabeled hormone required todisplace a given amount of labeled hormone could then be used toestimate mass of an sample hormone. Separation of the fractions whichwere unreacted with the antibody (unbound) was carried out by a varietyof chemical separation methods. In the original teaching of Yalow andBerson (1959), separation of the antibody-bound fractions of insulinfrom the unbound (free) fractions of insulin was carried byelectrophoresis. Subsequent to their report, many means of separatingbound from free fractions have been utilized, including columnchromatography, salt or organic solvent precipitation of the protein(antibody), double antibody (in which the gamma globulin fraction of thespecies immunized against the hormone is then introduced to a differentspecies to create an anti-antibody, or second antibody, and solid phase,in which the antibody is held by electrostatic forces to a solidinterphase such as the inner wall of a test tube, flat disc, or elongatestick (dipstick) and separation of bound from free requires simplephysical separation of the solid phase from the liquid phase containingthe free fractions. A variant of the technique of radioimmunoassayinvolved coupling small, non-immunogenic molecules to larger, highlyimmunogenic molecules, such as bovine serum albumen (BSA), thyroglobulin(TG), or keyhole limpet hemocyanin (KLH) and stimulation of antibodiesthat recognized the smaller, non-immunogenic, portion of the haptenmolecule. This modification of the technique permitted quantification ofsmall hormones, such as steroids and prostaglandins.

While radioimmunoassay is a very useful tool for conducting research andfor certain clinical applications, it has several drawbacks as far aspractical management of endocrine or other hormonal states. A majordrawback is the use of an antibody as a “capture protein.” Developmentof polyclonal antibodies is accomplished by administering the hormone toan animal that regards it as foreign and develops antibodies against it.The process is very much trial and error and involves the use of anumber of animals and screening of the antibody before determination ofits usefulness. Once an ideal polyclonal antibody preparation has beenobtained, the animal's plasma must be harvested and husbanded carefully,for once the animal dies, the supply of that particular antibody is lostforever.

The act of mounting an immune attack against a foreign protein andproducing antibodies is actually a mixture, or collection of antibodies(hence the term “polyclonal antibody”), each of which is directedagainst a particular amino acid sequence called an epitope. A refinementof this process involves the production of monoclonal antibodies.Monoclonal antibodies are derived by collecting individual spleen cellsfrom animals immunized by administration of a foreign protein andculturing the lymphocytes in vitro. The cells are then screened todetermine their binding characteristics, and those cells that possessappropriate binding are then cloned and maintained as anantibody-specific, continuous cell line. Thus, once appropriate cellcultures are obtained, they may be kept essentially indefinitely,thereby obviating one of the negative aspects of polyclonal antibodies.

However, monoclonal antibodies also have some drawbacks. For one thing,they are so specific as to be a detriment in some cases. Monoclonalantibodies are directed against amino acid sequences (epitopes) that areoften common features of the tertiary structure of proteins. In thiscase the monoclonal antibodies are really not specific as one mightbelieve at first. This drawback can be overcome by very stringentscreening and validation of the assays utilizing monoclonal antibodies,but greater effort is often required. Additionally, monoclonalantibodies tend to be monovalent, which may restrict hierarchical orsandwich type coupling to other molecules for the purpose of separationor of amplification of the reporter signal.

Immunoassays fall into two principal categories: “sandwich” and“competitive,” according to the nature of the antigen-antibody complexto be detected and the sequence of reactions required to produce thatcomplex. Generally, the sandwich immunoassay calls for mixing the samplethat may contain the analyte to be assayed with antibodies to theanalyte. These antibodies are mobile and typically are linked to adetectable label or a disclosing reagent, such as dyed latex or aradioisotope. This mixture is then applied to a chromatographic mediumcontaining a band or zone of immobilized antibodies to the analyte ofinterest. When the complex of the molecule to be assayed and the labeledantibody reaches the zone of the immobilized antibodies on thechromatographic medium, binding occurs and the bound labeled antibodiesare localized at the zone. This indicates the presence of the moleculeto be assayed. This technique can be used to obtain quantitative orsemi-quantitative results. Examples of sandwich immunoassays performedon test strips are described by U.S. Pat. No. 4,168,146 to Grubb et al.and U.S. Pat. No. 4,366,241 to Tom et al., both of which areincorporated herein by reference.

In competitive immunoassays, the label is typically a labeled analyte oranalyte analogue which competes for binding of an antibody with anyunlabeled analyte present in the sample. Competitive immunoassays aretypically used for detection of analytes such as haptens, each haptenbeing monovalent and capable of binding only one antibody molecule.Examples of competitive immunoassay devices are those disclosed by U.S.Pat. No. 4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta,and U.S. Pat. No. 5,208,535 to Buechler et al., all of which areincorporated herein by reference.

Although useful, currently available immunoassay techniques have anumber of disadvantages. For example, because antibodies and otherimmunoassay reagents are susceptible to environmental conditions,at-home or on-site methods are problematic. Further, antibodies continueto be expensive to produce. Accordingly, it would be advantageous toemploy an analyte assay system with all of the advantages of animmunoassay, but which is free of the inherent disadvantagestraditionally associated with such immunologically based systems.

In addition to immunoassays, protein-binding assays have been utilizedto quantitatively detect various analytes within a sample. These assaysystems utilize a protein, such as a protein receptor, which is specificfor a particular analyte target. Unfortunately, because these systemsutilize the entire protein and the protein may have binding sites formore than one target analyte, there can be problems withcross-reactivity and assay accuracy. For example, in angiotensin II SPAreceptor binding assays, the whole angiotensin membrane protein receptoris immobilized on a bead. This bead-receptor complex is then contactedwith a sample, binding any angiotensin present in the sample.

Determination of optimum breeding time is important to the success ofbreeding domestic species. For example, the ability to rapidly andaccurately measure equine ovarian steroid hormone estradiol (E2), in thefield, would greatly benefit veterinarians in assisting managers ofequine breeding farms in making breeding management decisions. Theliterature is replete with reference to early pregnancy failure in avariety of domestic species, e.g. sheep, 20 to 30% (Edey, 1969; Hanley,1961; Nancarrow, 1994), goats, 6 to 42% (Kidder et al., 1954; Diskin etal., 1980), pigs, 20 to 30% (Bolet, 1986), cattle, 8 to 42% (Pope etal., 1985) and mares, 15 to 25% (Ginther, 1986; Villahoz et al., 1985;Ball et al., 1986; Ball et al., 1987). A major component of these lossesrepresents errors of fertilization and/or exchange of geneticinformation (Hunter, 1994; King, 1990) or errors in the interactionbetween the maternal uterine unit and the developing conceptus (Bazer etal., 1986) even if successful breeding has occurred. With such lossesinherent to the reproductive process, it is critical for veterinariansand managers to select the optimal time for breeding to maximize thepotential for a successful pregnancy.

Current on-site methods for selecting the optimal time of breeding inthe equine industry include the observation of behavioral interactionsbetween the mare and stallion (teasing), rectal palpation and/orultrasound to monitor ovarian follicular growth, as well as obligatorybreed registry regulations. Teasing refers to the observation of themare's behavior to the presence of a stallion. Distinct behavioralpatterns are observed during each phase of the equine estrous cycle(Ginther, 1992). Palpation involves the insertion of the arm into themare's rectum to manually determine ovarian follicular activity. Often,ultrasound viewing of ovarian follicular activity is also utilized.Accurate record of teasing and palpation/ultrasound data are essentialfor good breeding management.

Predicting optimal breeding time is also important because the nature ofthe breeding business adds constraints to successful conception. Mostbreeders do not keep stallions on their farms, and access to popularstallions requires scheduling and transportation of the mare to thestallion at a predetermined time (reserved booking date/time). Thisprocess adds substantial cost and financial risk and, therefore,increases the value of a tool that can predict ovulation. Furthermore,the arbitrary January 1 birth date employed by many breed registriesrequires that breeding and pregnancy occur as early in the breedingseason as possible. Given a gestation length of 340 days, on average(Jeffcoat, 1972), early pregnancy results in birth as early in thefollowing January as possible, creating a more mature, market valuable,horse for sale or training. According to the TBH Market Watch (1997),January foals were sold for 25% more compared to foals born in othermonths (i.e., February through July).

While predicting optimal breeding time is advantageous, few managershave the skill to accurately perform rectal palpation and even fewerhave access to ultrasound. Veterinarians customarily charge managers foreach farm visit and for each mare which they have to palpate and/orultrasound. Moreover, traditional on-site breeding management practices(teasing, rectal palpation and/or ultrasound) cannot determine if apre-ovulatory sized follicle will ovulate or regress. Accordingly, anytechnology which would allow the pre-selection of mares which requireveterinary attention would be more efficient and economical.

The ovarian steroid hormone estradiol (E2) is a reliable indicator ofthe time of ovulation in mammals. During the breeding season, E2 is acritical hormone for normal follicular maturation, uterine endometrialdevelopment and ovulation in mares, as well as other mammals (Lipner,1988). Furthermore, E2 exhibits a distinct secretory pattern during theestrous cycle during the breeding season. Mares are seasonal breederswith an annual reproductive cycle consisting of four phases: thebreeding season (late spring to summer), the autumnal transition toAnestrus (late summer and fall), Anestrus (winter) and the vernaltransition from Anestrus to the breeding season (late winter and spring)(Sharp, 1980). The breeding season is characterized by repeated estrouscycles, providing multiple, successive opportunities to become pregnant.Concentrations of E2 in blood are low (5 to 10 pg/ml) during thediestrus period; increase dramatically (30 to 70 pg/ml) in the 3 to 4days preceding ovulation; peak 1 day prior to ovulation, on average;then decline to low diestrus concentrations (Pattison et al., 1974).Estrous cyclicity continues until either pregnancy results or decliningday length initiates the transition into anestrus. Autumnal transitionis not very well characterized. Briefly, a decline inhypothalamic-pituitary support, including GnRH (Strauss et al., 1979),LH and FSH secretion (Ginther, 1992), results in a progressivedisruption in ovarian follicular growth and steroid hormone production.Anestrus begins when GnRH secretion reaches low, unvarying levels, LHand FSH secretion cease and ovarian follicular activity ceases (Ginther,1992). Increasing day length in late winter initiates an increase inGnRH secretion and FSH secretion resulting in increased folliculargrowth (Ginther, 1992). An average of between 3 and 4 large,pre-ovulatory size follicles develop but fail to ovulate during thisperiod (Tucker et al., 1993). These large, anovulatory follicles createconsiderable confusion as they are similar to ovulatory follicles indiameter, but they are unaccompanied by an increase in LH and they donot ovulate. Furthermore, these anovulatory follicles do not produce E2.LH secretion and E2 secretion do not recommence until immediately priorto the first ovulation of the year (the start of the breeding season)(Sharp et al., 1997).

Currently, veterinarians and breeding managers can determine E2 levelsin mares only by submitting blood samples to a diagnostic laboratory.This process is costly and results are usually not available for 24hours to one week, depending on the lab. Present commercial assaysystems for E2 require several hours of incubation and expensivedetection systems. Such assays utilize radioactive substances orhazardous chemicals and complicated procedures, neither of which iscompatible with on-site use. For example, U.S. Pat. No. 5,460,976teaches an luminescence assay for measuring oestradiol in the bloodsample of an equine using two antibodies, an antibody against humanoestradiol and an antibody against human FSH. Unfortunately, in view ofthe extreme sensitivity of the immunologic components to bothenvironmental condition and chemical environment, this method also doesnot lend itself to on-site use. Accordingly, the ability to rapidly andaccurately measure E2, in the field, would greatly benefit veterinariansin assisting managers of equine breeding farms in making breedingmanagement decisions.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns methods and materials for accuratelyassessing the presence or absence of an analyte of interest in a sample.The LBD is then used to detect the presence of a target analyte in asample based on binding of the analyte by the LBD. In one embodiment,the subject invention involves utilizing a ligand binding domain (LBD)of a receptor to selectively capture a target analyte that is bound bythe LBD. Accordingly, the methods of the invention can be used to detectany target analyte for which there is a receptor molecule having aligand binding domain that specifically binds the target analyte. In oneembodiment, the receptor molecule is a protein or polypeptide.

An exemplified embodiment of the present invention is directed tomethods and materials for the rapid detection and quantitation ofovarian steroid hormone estradiol (E2) in the peripheral circulation ofmammals, particularly horses. In this embodiment, the LBD utilized is arecombinantly-expressed polypeptide derived from the native equineestrogen receptor (eER) capable of specifically binding E2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows estradiol concentrations (pg/ml) in mares during twoconsecutive estrous cycles.

FIG. 2 shows estradiol concentrations (pg/ml) in mares during the vernaltransition into the breeding season.

FIGS. 3A–3F show components and steps of a liquid phase assay of thepresent invention, wherein estradiol is the analyte of interest.

FIGS. 4A–4E show components and steps of a solid phase assay of thepresent invention, wherein estradiol is the analyte of interest.

FIGS. 5A–5C show components and steps of a lateral flow-based assay ofthe present invention, wherein estrogen is the analyte of interest.

BRIEF DESCRIPTION OF SEQUENCE SEQ ID NO:1 is an amino acid sequence ofan equine estrogen receptor ligand binding domain according to thepresent invention. DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns methods and materials for accuratelyassessing the presence or absence of an analyte of interest in a sample,particularly in physiological samples. The methods of the inventionutilize a ligand binding domain (LBD) of a receptor molecule that iscapable of binding to a target analyte to selectively capture theanalyte target specific for that LBD. In one embodiment, the receptormolecule is a protein or polypeptide. The ligand binding domain isallowed to react with a sample and the presence or amount of ligand(i.e., target analyte) bound by the LBD is determined. The subjectinvention includes within its scope any method or assay in which bindingof an LBD to a target analyte can be used for detection or quantitationof the analyte.

The subject invention also concerns the ligand binding domain of theequine estrogen receptor. In an exemplified embodiment, the ligandbinding domain has an amino acid sequence of SEQ ID NO:1, or a fragmentor variant thereof that retains substantially the same ligand bindingaffinity as that associated with the polypeptide of SEQ ID NO:1. Thesubject invention also concerns polynucleotide sequences that encode aligand binding domain of the present invention.

In one embodiment of the present methods, an LBD is attached to a solidsupport and contacted with a sample to be assayed for the presence orlevels of a target analyte that the LBD specifically binds. Afterwashing, a conjugate comprising target analyte conjugated with adetectable marker, such as an enzyme or radiolabel, is incubated withthe LBD on the solid support. Levels of the conjugate bound to the LBDon the support are then determined.

In another embodiment, a sample to be assayed for the presence or levelof a target analyte is contacted with a conjugate comprising an LBD forthe analyte to be assayed conjugated to a detectable marker, such as anenzyme or a radiolabel. The sample is then contacted with target analyteimmobilized to a support matrix, such as SEPHAROSE. After washing, theamount of the LBD-marker conjugate bound to the target analyte on thesupport matrix is determined.

In a further embodiment, a lateral flow based assay is provided in thepresent invention for the detection of a target analyte. A samplecontaining a target analyte of interest is added to an application wellin a lateral flow device. The test sample flows toward a zone containinga solid support moiety that has been coated with a ligand-bindingdomain. When the sample front reaches this zone, the ligand-bindingdomain attached to the solid support moiety is released from the pad onwhich they are bound and then allowed to interact with target analytethat may be present in the test sample. Target analyte in the samplebinds to the solid support bound LBD and flow continues toward a targetanalyte affinity matrix. All solid support moieties that possess“unoccupied” LBD are captured by the target analyte affinity matrix,whereas all solid support moieties having fully-occupied LBD willcontinue toward the capture zone where they are trapped. Results can beassessed visually by colorometric means with the intensity of colorbeing directly proportional to target analyte concentration within thetest sample.

The present invention provides a novel approach to analytical methods byutilizing a “capture protein” prepared by genetic engineering of aligand binding domain of a naturally occurring receptor molecule for atarget analyte. The protein from which the LBD is derived has an aminoacid sequence which is unique and binds the target analyte specifically.In an exemplified embodiment of an LBD of the present invention, aligand binding domain portion of a receptor gene for E2 was produced bygenerating a cDNA fragment coding for the eER ligand binding domain(corresponding to amino acids 301–564 of the full-length receptorprotein) by PCR utilizing the eER cDNA plasmid as template. PCR primerswhich flank the LBD of the eER were designed and synthesized by GeminiBiotech, Ltd. These primers also created XmaI restriction sites forsubcloning of cDNA into a pBAD expression vector. Plasmid cDNA wasisolated with QIAprep Spin DNA purification columns (Qiagen) andpresence of the correct insert size was confirmed by restrictionanalysis. Following isolation of the eER-LBD subclone, the entire LBDcoding region was sequenced to confirm no errors were incorporated byPCR amplification. The eER-LBD peptide was overexpressed in E. coliJM103 cells (available from ATCC, Rockville, Md.) transformed with thepBAD-23-eER-LBD expression vector. Purity of the protein was assessed bySDS-PAGE and Western blot and/or ligand blot analysis.

Use of a genetically engineered LBD as a “capture protein” has severaladvantages over existing systems. First, the ligand binding domain isspecific to the hormone as it represents the naturally occurring tissuereceptor mechanisms for recognizing a hormone. Second, use of the ligandbinding domain as a “capture protein” is advantageous because the aminoacid sequence can be altered by making point mutations in theoligonucleotide primers, and the properties of the ligand binding domaincan then be adjusted to suit the needs of an assay system, or othersystems requiring hormone-receptor recognition. Third, the in vitroexpression of a ligand binding domain assures that a consistent,repeatable source of “capture protein” will be available. Fourth, theligand binding domain can also be altered to accept organic linker armsfor the purpose of attachment to solid phase, or to create attachmentpoints for reporter molecules, such as enzymes, fluorescent molecules orother moities that can indicate the presence of the ligand bindingdomain having bound target analyte.

While a wide variety of analytes may be assessed utilizing the methodsof the invention, the present disclosure exemplifies methods andmaterials of the invention for the detection of ovarian steroid hormoneestradiol (E2). It should be understood, however, that the subjectinvention is not limited to the detection of estrogens and estradiol.Analytes which can be detected using the methods of the presentinvention include all those for which a ligand binding domain (LBD) canbe derived.

Estradiol levels in mares increase significantly in blood just prior tothe first ovulation of the year and just prior to each subsequentovulation, as shown in FIG. 1. Traditional breeding management practices(teasing, rectal palpation and/or ultrasound) cannot determine if apre-ovulatory size follicle will ovulate or regress. During vernaltransition, E2 synthesis and secretion is very low or absent until thefirst preovulatory follicle of the year develops, where upon E2concentrations in blood increase (30 to 70 pg/ml) dramatically (Davis etal., 1990), as shown in FIG. 2.

An exemplified embodiment of the present invention provides methods forpersons such as veterinarians and breeding managers to determine if afollicle is a “vernal transition (non-ovulatory) follicle,” or if thebreeding season has begun (i.e., a fertile ovulation can be expected)and thereby make breeding management decisions accordingly. Componentsfor the present methods are shown in FIGS. 3A, 4A and 5A, and included aligand-binding domain (LBD) of the equine estrogen receptor (eER).Sequence analysis of the LBD of the eER (nucleotide sequence offull-length eER gene: Genbank Assession #AF124093) shows 90% identityand 95% similarity to other mammalian ER-LBDs, as shown in Table 1.

TABLE 1 Comparison of the equine estrogen receptor nucleotide (AF124093)and deduced amino acid sequences to those of other species Full-lengthEstrogen Receptor Ligand Binding Domain GenBank Nucleotide Amino AcidAmino Acid Amino Acid Amino Acid Specie Accession Homology IdentitySimilarity Identity Similarity Human 182192 89% 89% 93% 90.5% 94.9%Mouse 193180 87% 87% 91% 90.5% 94.9% Rat 56120 83% 86% 90% 90.2% 94.3%Pig 587554 91% 91% 94% 90.5% 94.6% Cow 1575521 ND ND ND 95.0% 96.0%Sheep 1617201 89% 90% 94% 90.5% 94.9% Chicken 63380 78% 76% 84% 85.5%91.6%

Published data of all amino acids critical for E2 binding to the ER-LBDin other species (Ekena et al., 1996) are in identical sequencepositions in the eER-LBD (Gly⁵²¹, His⁵²⁴, Leu⁵²⁵, Met⁵²⁸). Therefore,the binding kinetics of the eER-LBD should not be different from datapublished for other species (K_(d)=0.1 nM)(Ekena et al., 1996). SuchER-LBD peptides can be expressed as individual peptides (e.g., residues301 to 564 of eER) and maintain their specificity for E2 (Wrenn et al.,1993; Ekena et al., 1996).

Hence, it should be readily apparent by those of ordinary skill in theart that the inherent homology in the estrogen receptor nucleotidebetween mammalian species extends the utility of the subject inventionto the diagnostic detection of E2 within mammalian species other thanhorses. For example, using either a modified or unmodified ER-LBD, thepresent invention is applicable to detection of ovulation in humans aswell, particularly since preovulatory profiles of estrogen in women areeven more robust than those in mares.

There are several alternative embodiments of the methods of the presentinvention that utilize a recombinantly-expressed polypeptide whichcontains a ligand binding domain (LBD) that can bind an analyte ofinterest. One embodiment concerns a liquid phase assay, the componentsfor which are shown in FIG. 3A. A second embodiment concerns a solidphase assay, the components for which are shown in FIG. 4A. A thirdembodiment concerns a lateral flow assay, the components for which areshown in FIG. 5A. For the purpose of example, and as exemplified in thefigures, the analyte of interest can be E2, in which case, a polypeptidecontaining the LBD of the estradiol receptor is used.

An exemplified embodiment of the methods of the present inventionconcerns a liquid phase-based assay for equine E2 (FIGS. 3B–3F). In thefirst step, a sample, for example, blood from a mare to be tested isadded to a titrated amount of an eER-LBD enzyme conjugate (hereafterreferred to as “conjugate”). The conjugate is composed of arecombinantly-expressed LBD of equine estrogen receptor conjugated to acolorimetric enzyme, such as alkaline phosphatase or horseradishperoxidase. Conjugation of the eER-LBD with the colorimetric enzyme canbe accomplished using standard methods. Preferably, the conjugate istitrated such that, when incubated with the sample, it will bind amaximum concentration of 30 pg/ml of E2, becoming saturated. The secondstep of the method consists of incubation to allow ligand binding. Themixture of conjugate and serum is then added to an estrogen affinitymatrix. The estrogen affinity matrix functions as an estradiol affinityadsorbent, preferably consisting of a titrated amount of E2 immobilizedupon SEPHAROSE (Amersham Pharmacia). The affinity matrix can be preparedby the linkage of estrogen with SEPHAROSE as described by Greene et al.(1980). Briefly, 17α-allylestradiol 3-acetate is prepared by thereaction of estrone with allylmagnesium chloride followed by acetylationof the phenolic group. The 17α-allylestradiol 3-acetate is thenconverted to the side-chain epoxide by treatment withm-chloroperoxybenzoic acid and then reacted with reduced2-hydroxy-3-mercapto-n-propyl-SEPHAROSE-6B. Unreacted sulfhydryl groupsin the thiopropyl-SEPHAROSE will be blocked by treatment withiodoacetamide.

The affinity matrix is then washed and conjugate which has not bound toE2 within the sample will bind to the immobilized E2 on the affinitymatrix and be “captured.” However, conjugate which has bound E2 from thesample will flow through the matrix and, hence, not be captured. A colorsubstrate can then be added to the estrogen affinity matrix, binding tothe colorimetric enzyme component of any conjugate which has bound tothe immobilized E2, as illustrated in FIG. 3. The intensity of the colorwill depend on the amount of complex “captured.” Hence, colordevelopment occurs in a manner inversely proportional to analyte (E2)concentration within the sample. For example, if the mare's serumcontains low concentrations of E2, an intense color will result, as mostor all of the complexes will be captured on the matrix. However, ifgreater than 30 pg/ml of E2 are in the mare's serum, no color willresult, as all the complexes will pass through the matrix withoutbinding to the immobilized E2. While any number of color stages may beused, for unambiguous visualization it is preferable that the conjugatebe titrated in such a way that there is a three stage color possibility.For example, where the sample is mare's serum and the analyte ofinterest is E2, intense color indicates low E2, light color indicatesbetween 5 and 25 pg/ml of E2 and no color indicates greater than 30pg/ml E2.

While the color development is inversely proportional to theconcentration of the analyte (17β-estradiol) in the test sample, toensure color reactions that are visible to the eye, it should beunderstood that amplification of the colorimetric signal can beaccomplished by using commercially available antibodies to horseradishperoxidase (HP) and alkaline phosphatase (AP). These antibodies can bebiotinylated and amplification achieved with enzyme-labeledstreptavidin. Alternatively, a sandwich assay which utilizesstreptavidin and biotinylated enzyme can be incorporated to achievemaximal amplification.

The E2 can be immobilized onto the surface of the affinity supportmatrix by any method that affixes the E2 to the support in asubstantially irreversible manner, such as where the E2 is covalentlybound to the support matrix. Further, E2 can be attached or coupled tonumerous other support matrices known in the art using standard methods.Suitable solid-phase support matrices can be composed of nitrocellulose,DEAE, glass, nylon, particulate silica, polystyrene, polyethylene,polyamides, polyacrylamides, polyvinyls, polypropylene, celluloseagarose, dextran or any other suitable material known in the art. Thesolid support matrix can be in the form of a vessel, a chamber, adipstick, beads, particles, membranes, or other forms known in the art.Suitable membranes include those composed of nylon, nitrocellulose orpolyvinylidenedifluoride (PVDF).

Because many color substrates are commercially available for eachcolorimetric enzyme and each has its own optimal conditions for colordevelopment, there are a variety of color substrates which can be usedwithin the methods of the present invention. Examples include, but arenot limited to, p-Nitrophenyl Phosphate (PNPP; yellow), Fast Red (red)and 5-Bromo-4-Chloro-3′-Indolyphosphate (BCIP)/Nitro Blue TetrazoliumChloride (NBT; black-purple) for the AP conjugates; and, 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS; green),3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB; blue or yellow) and3,3′-diaminobenzidine (DAB; brown) for the HP conjugates at theconcentrations recommended by their supplier (Sigma Chemical Companyand/or Pierce Chemical Company). The substrate which yields the mostdistinguishable changes in color in parallel to changes in analyteconcentrations should be selected. However, stability of the substrateprior to the reaction, solubility of the product and sensitivity willall be considerations for substrate selection as well.

The methods of the present invention are also exemplified by a solidphase assay for detecting an analyte, such as E2 in an female equine(FIGS. 4B–4E). In this embodiment, a titrated concentration of eER-LBDis immobilized onto a support matrix, such as a nylon membrane,dipstick, coated vessel or filtration chamber. The solid support matrixcan be composed of any of the materials described for the E2 affinitysupport matrix. As with the liquid phase embodiment, severalconcentrations of eER-LBD can be used. Preferably, the optimum titrationwill be that which is saturated by 30 pg/ml 17β-estradiol.

In the first step of the solid phase embodiment, immobilized (solidphase) eER-LBD is incubated at room temperature with the sample to allowfor binding of eER-LBD with 17β-estradiol present in the sample. In thesecond step of the solid phase embodiment, the sample is then passed ordrawn through or evacuated by vacuum filtration through the solidsupport matrix and the support matrix washed. Optionally, the washsolution can be a buffer that contains a blocking agent (e.g., gelatinor BSA) to reduce nonspecific binding. After the washing/blocking step,a titrated concentration of a 17β-estradiol-enzyme conjugate (hereafterreferred to as the estrogen conjugate) is added to the membrane andincubated to allow interaction with the solid-phase eER-LBD that has notbound ligand. As with the liquid phase embodiment, the enzyme of theestrogen conjugate can be a colorimetric enzyme. After incubation,estrogen conjugate which is not bound by eER-LBD is removed and thesolid support matrix can be washed. The solid support is then reactedwith a substrate for the given enzyme conjugate as previously describedin the liquid phase embodiment. The amount of colorimetric enzyme whichremains on the solid support is inversely proportional to the amount of17β-estradiol in the sample. It is preferable to titer the amount ofsolid-phase eER-LBD in such a way that a three-stage color developmentscheme will occur as described in the liquid phase embodiment. Inaddition, although the above described method utilizes a vacuumfiltration manifold, the method can be easily adapted to other systems,such as a gravity flow system.

It should be appreciated that a ligand binding domain used in themethods of the present invention can be modified so as to adjustspecificity and/or affinity to suit diagnostic needs. For example, alinker arm may be added to the LBD of the mammalian estrogen receptorfor conjugation to plastic, enzymes, etc. In addition, the amino acidsubstituents of the LBD can be modified so as to alter the affinity ofthe LBD for its associated analyte. For example, mutations in the aminoacid sequence of the LBD, such as amino acid substitutions, deletionsand/or additions, are contemplated by the present invention.

Further, it should be understood by the ordinarily skilled artisan thatthe present invention may be further modified to resemble an “antibodysandwich” assay. For the purposes of this disclosure, the term “antibodysandwich” assay simply means an assay in which the analyte to bedetermined is “sandwiched” by an immunochemical reaction between a solidsurface treated with a first antibody reactive with the analyte to bedetermined and the same or a different second antibody which has beencoupled to an enzyme label. This “antibody variant” of the subjectinvention differs from traditional antibody sandwich assays in that thevariant uses one antibody and the LBD of the subject invention, whereastraditional sandwich assays utilize two antibodies. For example, in theaforementioned liquid phase embodiment, a titrated amount of antibodiesspecific for the eER-LBD are immobilized upon the SEPHAROSE (AmershamPharmacia) solid support, in lieu of 17β-estradiol. Alternatively, inthe solid phase embodiment, an antibody-enzyme conjugate is addedinstead of the 17β-estradiol-enzyme conjugate. In this variant of thesolid phase embodiment, the antibody is specific for the eER-LBD. Inboth of these antibody-variants of the liquid and solid phaseembodiments, the amount of antibody used is titrated as previouslydescribed regarding the eER-LBD. Further, monoclonal antibodies may beused in the assays, as disclosed in U.S. Pat. No. 4,376,110.

Additionally, U.S. Pat. No. 4,228,240 describes the stabilization ofperoxidase containing compositions for use in enzyme immunoassay kits.U.S. Pat. No. 4,931,385 discloses an improved blocking solution whichprotects against nonspecific antibody binding and an improvedantibody-enzyme conjugate which protects the antibody from loss ofreactivity and immunological binding specificity even if the reagentshad been subjected to hot, humid environmental conditions. Such reagentsmay be used in connection with the subject invention.

The methods of the invention can be used to detect any target analytefor which there is a protein that binds to the analyte and for which aligand binding domain can be derived. Target analytes include analytessuch as hormones, enzymes, lipoproteins, bacterial or viral antigens,immunoglobulins, lymphokines, cytokines, drugs, soluble cancer antigens,and the like. These analytes include various proteins such asprotamines, histones, phosphorylated proteins, nucleoproteins, such as,for example, transcortin, erythropoietin, transferrin, variousglobulins, thyroxin-binding globulin, the immunoglobulins of varioussubclasses (IgA, IgG, IgD, IgE, and IgM), various complement factors,and blood clotting factors such as fibrinogen, Factor VIII, tissuethromboplastin, and thrombin. Further, the relationship of the bindingpair (i.e., the target analyte and the protein from which the LBD isderived) is not limited. For example, the relationship may be one ofenzyme-substrate, enzyme-inhibitor, enzyme-co-enzyme, etc. In additionto E2, other steroids including, but not limited to, progesterone andtestosterone, and other hormones such as insulin, glucagon, relaxin,thyrotropin, somatotropin, luteinizing hormone, follicle-stimulatinghormone, gastrin, bradykinin, vasopressin, and various releasing factorsare suitable analytes. A wide range of antigenic polysaccharides canalso be determined such as those from Chlamydia, Neisseria gonorrheae,Pasteurella pestis, Shigella dysentereae, and fungi such as Mycosporumand Aspergillus. Another major group comprises oligonucleotide sequenceswhich react specifically with protein targets.

The test sample can be any material suspected of containing the analyteof interest. The sample can be derived from any source, such asphysiological fluid, including blood, saliva, sweat, urine, milk,mucous, etc. The sample can be used as obtained directly from the sourceor following a pretreatment so as to modify its character. Pretreatmentmay involve separating plasma from blood, diluting viscous fluids, orthe like. Methods of treatment can involve filtration, distillation,concentration, inactivation of interfering components, and the additionof reagents. For example, the test sample may be dissolved in orsupplemented by a buffer to provide a suitable medium for theincubations of the invention. Further, in the case of the antibodyvariants of the subject invention, an additive may be included tofacilitate immunologic reactions involving the antibody.

A further embodiment of the methods of the subject invention utilizes amodification to the lateral flow technique described in U.S. Pat. Nos.4,943,522; 5,766,961; and 5,770,460. In the first step of thisembodiment, a biological sample containing the target analyte ofinterest (e.g., estrogen) is added to an application well in a lateralflow device. Lateral flow is accomplished by incorporating anon-bibulous support, with inherent hydrophobic properties, whichfacilitates non-bibulous lateral flow of the test sample to variouszones. The test sample flows toward a zone containing colored latexparticles that have been coated with eER-LBD. The conjugation of theeER-LBD to latex particles can be by passive adsorption or via acovalent linkage. When the sample front reaches this zone, the coloredlatex particles are released from the pad from which they areimpregnated and allowed to interact with the estrogen (analyte) presentin the test sample. Estrogen in the sample binds to the latex boundeER-LBD and flow continues toward an estrogen affinity matrix. All latexparticles that possess “unoccupied” eER-LBD are captured by the estrogenadsorbent, while all latex particles with fully-occupied eER-LBD willcontinue toward the capture zone where they are trapped in a verydefined area. The capture zone can utilize a number of high affinityreactions including streptavidin-biotin or antibody-antigeninteractions. Results are assessed visually with the number of capturedbeads, and thus intensity of color, being directly proportional toestrogen concentrations within the test sample (see FIG. 5A). Positivecolor development for physiological relevant concentrations of analyteis controlled by careful titration of the number of latex beads, thenumber of eER-LBD sites on each bead and/or the concentration ofaffinity adsorbent used.

It should be understood by those of ordinary skill in the art thatvarious diagnostic devices usable in connection with other bindingassays may be used in connection with the subject invention. Forexample, U.S. Pat. No. 4,361,537 and U.S. Pat. No. 4,855,240 disclosetest devices comprising a highly absorbent material capable oftransporting the test sample by capillarity. Further, certain devicesrely on improved transverse flow through a filter in order to removeparticulate and/or colored matter from the sample which may otherwiseinterfere with an accurate colorimetric reading, such as that disclosedin U.S. Pat. No. 4,623,461. It should also be appreciated by those ofordinary skill in the art that the present invention may be utilized ina device which uses a plurality of test elements, each for a differentanalyte, with all elements being supplied analyte from a single quantityof test sample, along different flow paths, as described in U.S. Pat.No. 4,323,536.

In addition, the eER-LBD of the present invention may be modified inorder to detect and measure phytoestrogen within mammals, particularlyagricultural animals such as horses and cows. Phytoestrogen is ofconcern to ranchers because phytoestrogen may accumulate in animals thateat plants with high amounts of phytoestrogen. Abnormal levels ofphytoestrogen may then result in abnormal changes in the animal'sestrous cycles.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Cloning of the Equine Estrogen Receptor

The full-length equine estrogen receptor (eER; alpha-type; clone KjmeER-13) was cloned from an estrous endometrial cDNA library (ZAPEXPRESS; Stratagene) using standard screening procedures (Sambrook etal., 1989). One microgram of eER cDNA plasmid was transformed into DH521competent cells and plated onto LB-agar. Two independent colonies werepicked and selectively grown in 200 ml of Luria-Bertani (LB) culturebroth with kanamycin (100 ug/ml ). Equine ER cDNA plasmid was isolatedfrom the growth cultures with the Plasmid Midi Kit (Qiagen). The cDNAinsert (>>4000 bp) was sequenced in its entirety by the DNA sequencingcore laboratory of the Interdisciplinary Center for BiotechnologyResearch (ICBR) at the University of Florida. Nucleotide sequencecomparisons were performed with the BLAST feature of the National Centerfor Biotechnology Information, as shown in Table 1. The eER nucleotidesequence was submitted to GenBank and assigned the accession numberAF124093.

EXAMPLE 2 PCR Cloning of the eER Ligand Binding Domain

A cDNA fragment coding for the eER ligand binding domain (LBD; aminoacids 301–564 based on the amino acid numbering of the full-lengthreceptor sequence at Accession No. GI:4325290) was generated by PCRutilizing th eER cDNA plasmid as template. Oligonucleotides that flankthe LBD of the estrogen receptor were designed and synthesized by GeminiBiotech, Ltd. and included XmaI sites at the 5′ ends for futuresubcloning steps. Following 30 cycles of PCR (95° C. for 1 minute, 55°C. for 2 minute, 72° C. for 2 minutes), 10 μl of the PCR reaction wasseparated on a 1.8% agarose gel to confirm amplification of the correctproduct size (840 bp). One μl of the PCR reaction was ligated to pCR 2.1cloning vector (Invitrogen) using the TA cloning principles. Theligation reaction (3 μl) was transformed into One Shot competent cells(Invitrogen) and plated onto LB-kanamycin agar plates that containedX-gal. Ten randomly selected recombinant (white) colonies were selected,inoculated into LB-ampicillin and grown overnight at 37° C. Plasmid cDNAwas isolated with QIAprep Spin DNA purification columns (Qiagen) andpresence of the correct insert size confirmed by restriction analysis.Following plasmid isolation, the eER-LBD clone was sequenced in itsentirety by the DNA sequencing core laboratory of the InterdisciplinaryCenter for Biotechnology Research (ICBR) at the University of Florida toconfirm no errors were incorporated by PCR amplification.

EXAMPLE 3 Subcloning into an Expression Vector

After confirming no errors were generated by PCR and that the codingsequence is in frame, the nucleotide sequence which codes for eER-LBDwas released from the pCR 2.1 vector by digesting with XmaI and gelpurifed. The eER-LBD coding sequence was then subcloned into a pBADexpression vector which had been modified to include a pectate lyasesecretion signal (pBADPL; Gemini Biotech, U.S. Pat. No. 5,576,195).Furthermore, the pBADPL vector added a 6X-histidine tag and atermination codon to the 3′ end of the subclone. Five μl of the ligationreaction was transformed into the E.coli strain JM103 (ATCC) that hadbeen rendered competent using CaCl₂ (Sambrook et al., 1989). After a 30minute incubation on ice, cells were heat shocked for 1 minute at 42°C.; then grown for 1 hour at 37° C. in SOC media with shaking (250 rpm).Transformation reactions were plated onto LB-ampicillin plates and grownovernight at 37° C. Twenty single recombinant colonies were inoculatedin LB medium containing 100 μg/ml ampicillin and grown overnight at 37°C. Plasmid DNA was isolated with QIAprep Spin DNA purification columnsand orientation determined by restriction analysis and/or DNAsequencing. Two clones, one in the correct and one in the reverseorientation (negative control), was utilized for protein expression.

EXAMPLE 4 Recombinant Expression of the eER-LBD Peptide

Initially, pilot studies were conducted to determine optimal conditionsfor induction. Single recombinant colonies for each clone were selectedfrom LB-ampicillin plates and inoculated into 2 ml of LB containing 100μg/ml ampicillin. Cultures were grown at 37° C. with shaking (250 rpm)to an OD₆₀₀=1–2. Five 10-ml aliquots of LB-ampicillin were eachinoculated with 100 μl of the overnight culture and grown at 37° C. withvigorous shaking to an OD₆₀₀=0.5 (mid-log phase). When an OD₆₀₀=0.5 wasobtained, 1 ml of each culture was removed and saved for future analysis(Time 0 h). To the remaining 9 ml of each of the five respective growthcultures, 90 μl of a 10-fold serial dilution of 20% L-arabinose(0.002%–20%) was added such that the final arabinose concentrationsranged from 0.00002%–0.2%. Cultures were grown an additional 4 hours at37° C. with shaking. One ml aliquots were removed (Time 4 h) andaliquots for both time points were centrifuged at maximum speed in amicrofuge for 30 seconds. The supernatant (excreted protein) and thecell pellet (protein secreted into the periplasmic space) were added toLaemmli sample buffer, fractionated on a 12% gel by SDS-PAGE and stainedwith Coomassie blue. Gels were examined to determine the optimalarabinose concentration for induction as well as the proportion of theprotein that is excreted into the medium. Once optimal conditions formaximum expression have been determined, expression can be scaled upaccordingly depending upon the yields and the needs.

EXAMPLE 5 Affinity Purification of the Recombinant eER-LBD Peptide

The culture media can be concentrated with Centricon-plus 80 centrifugalfiltration devices and dialyzed overnight against binding buffer (20 mMsodium phosphate, 500 mM sodium chloride, pH=7.8) at 4° C. Total proteinis determined with the BioRad protein assay. The eER-LBD protein isselected from the media by batch binding to ProBond resin (Invitrogen).An aliquot of the dialyzed media (equivalent to 5 mg of total protein)is brought to a total volume of 10 ml with binding buffer and dividedinto two 5-ml aliquots. One 5-ml aliquot is batch bound to 5 ml ofProBond resin with gentle rocking for 10 minutes at room temperature.The resin is settled by centrifugation at 800×g and the supernatantdecanted. This is repeated with the second 5-ml aliquot. The resin isthen washed three times with a native wash buffer (20 mM sodiumphosphate, 500 mM sodium chloride, pH=6.0) by resuspending the resin in10 ml of the wash buffer, rocking for 2 minutes and separating bycentrifugation at 800×g. After the final wash, the resin is transferredto a column. The eER-LBD protein is eluted from the resin byconsecutively adding 10 ml of each of four imidazole buffers (50 mM, 100mM, 250 mM, 500 mM), collecting 1 ml fractions and monitoring the OD₂₈₀of each fraction. Fractions with peak absorbance are pooled and purityis assessed by SDS-PAGE. Protein is quantified with the BioRad proteinassay after dialysis to remove the imidazole. Purity of the protein isassessed by SDS-PAGE and Western blot analysis.

EXAMPLE 6 Scatchard Analysis for Estrogen Binding to eER-LBD

After expression and purification, the equilibrium binding affinity ofeER-LBD for [³H]17β-estradiol can be determined by saturation analysis.Receptor preparations are diluted in TEDG buffer (10 mM Tris, 1.5 mMEDTA, 1 mM dithiothreitol, 10% vol/vol glycerol; pH 7.8). Dilutions (1–2nM) of the receptor preparation are then incubated at 4 C overnight withincreasing concentrations (0.2–20 nM) of [³H]17β-estradiol. Nonspecificbinding is determined in the presence of a 200-fold excess of unlabeled17β-estradiol. Free ligand is separated from bound ligand by addition ofan equal volume of dextran-coated charcoal slurry (1% charcoal, 0.01%dextran in TEDG). After a 10 minute incubation on ice, the charcoal ispelleted by centrifugation for 5 minutes at 14,000 rpm in a microfuge.The supernatent is carefully decanted and a portion used to quantitate[³H]17β-estradiol binding by liquid scintillation counting usingSCINTIVERSE cocktail. All data is then transformed by the method ofScatchard (1949) and an equilibrium dissociation constant (K_(d))determined for the eER-LBD. The calculated K_(d) value can compare tothose of the ER-LBD expressed in other species and by other expressionsystems.

EXAMPLE 7 Dissociation Kinetics of Estradiol from eER-LBD

The dissociation of [³H]17β-estradiol from the eER-LBD peptide can bemeasured by the exchange of [³H]17β-estradiol with an excess ofunlabeled 17β-estradiol. The eER-LBD peptide (1–2 nM) is incubated withsaturating (10 nM) concentrations of [³H]17β-estradiol at 4° C.overnight. Samples are then pre-incubated in a 29° C. waterbath for 30minutes prior to the addition of a 1000-fold excess of unlabeled17β-estradiol. Dissociation is allowed to progress at 29° C. andaliquots are removed every 30 minutes for 8 hours. Nonspecific bindingis determined by performing the overnight incubation in the presence ofa 200-fold excess of unlabeled 17β-estradiol. Dissociation-rateexperiments are terminated by DEAE filtration as described bySalomonsson et al. (1993). Briefly, DEAE paper discs (DE 81, WhatmanInternational Ltd) are put into a filtration manifold and the sample isapplied to the dry disc. A 2 minute incubation is performed prior tovacuum filtration to allow the eER-LBD-estrogen complex to bind to thefilter disc. After vacuum is applied, the discs are washed with 10volumes of ice-cold Tris buffer (pH 7.8) and transferred toscintillation vials. Radioactivity is allowed to dissolve in thescintillation cocktail for 4 hours before quantitation. Data is thenpresented as the amount of [³H]17β-estradiol displaced (percent of theinitial binding at time 0) as a function of time.

EXAMPLE 8 Stability of the Unoccupied eER-LBD

The stability of the eER-LBD can be determined by measuring the amountof specific [3H]17β-estradiol binding observed after increasingincubation times at 0° C. or 25° C. After a pre-incubation at either 0°C. or 25° C. for 2–24 hours, specific binding is determined as describedabove. Results are presented as the percentage specific binding relativeto the initial binding capacity as a function of time.

EXAMPLE 9 Conjugation of eER-LBD with Colorimetric Enzymes

The eER-LBD peptide can be conjugated with alkaline phosphatase (AP) orhorseradish peroxidase (HP) using preactivated enzymes and the EZ-Linkconjugation kits according to the manufacturer's recommendations (PierceChemical Company). For AP conjugation, maleimide activated AP is reactedwith free sulfhydryl (—SH) groups present in the eER-LBD peptide to forma stable thiol ether linkage (Ishikawa et al., 1983). The AP-eER-LBDconjugate is purified by gel filtration chromatography, adjusted to aprotein concentration equivalent to 1 nM eER-LBD and utilized forbinding studies. For HP conjugation, periodate activated HP is reactedwith amine (—NH₂) residues present in the eER-LBD peptide to form acovalent amide bond (Imagawa et al., 1982). After conjugation, thelinkage is reduced and the activated HP will be quenched withethanolamine. The HP-eER-LBD conjugate will be purified on a desaltingcolumn, adjusted to a protein concentration equivalent to 1 nM eER-LBD.

EXAMPLE 10 Conjugation of 17β-estradiol with Colorimetric Enzymes

The conjugation of 17β-estradiol with either HP or AP can beaccomplished a number of different ways including the mixed anhydridemethod (Munro et al., 1984), the carbodiimide method and the modifiedcarbodiimide method, which uses an activated estradiol ester preparedwith N-hydroxysuccinimide (Munro et al., 1988). Preferably, the mixedanhydrid method is utilized. Briefly, a derivative (hemisuccinate orcarboxymethyloxime) of 17β-estradiol (Steraloids) andsec-butylchlorocarbonate are dissolved in N,N-dimethylformamide at 0° C.N-methylmorpholine is added to remove hydrochloric acid and form themixed anhydride. In a separate reaction, enzyme (AP or HP) is dissolvedin water and N,N-dimethylformamide is added. The steroid solution thatcontains the mixed anhydride is gradually added to the enzyme solutionat 0° C. The reaction mixture is stirred for 60 minutes at −20° C. thenan additional 120 minutes at 0° C. After the incubation, sodiumbicarbonate is added and the reaction mixture is dialyzed overnightagainst distilled water at 4° C. The dialysate is passed over a SephadexG-25 column and the 17β-estradiol-enzyme conjugate is aliquoted andstored at −20° C.

EXAMPLE 11 Conjugation of Domain F of eER-LBD with Colorimetric Enzymes

A specific conjugation site can be added to the carboxy-terminus of theexpressed eER-LBD peptide (Pierce). This is not as desirable, however,since conjugation is 1:1 (enzyme:eER-LBD). Since the expressed peptidecan contain not only the LBD (domain E) of the ER but also domain F,tagging the carboxy-terminus does not impede ligand-receptorinteractions since crystallographic studies have shown this region isnot an integral part of the binding pocket of the estrogen receptor(Tanenbaum et al., 1998).

EXAMPLE 12 Coating Latex Particles with eER-LBD by Passive Adsorption

The recombinant eER-LBD is coated onto surfactant-free polystyrene latexparticles (Interfacial Dynamics Corp.) via physical adsorption. Mostcommercially available latex particles are hydrophobic and, thus, willadsorb proteins strongly and irreversibly via the hydrophobic domains inthe proteins. Briefly, 2.5 ml or 1.25 ml of a 4% or 8% solids,respectively, suspension of colored latex particles (e.g. those withsulfate or carboxyl surface functional groups) is diluted to 10 ml with25 mM 2-[N-Morpholino]ethanesulfonic acid, pH=6.0 (MES). The suspensionis then centrifuged at 3,000×g for 20 minutes to sediment the latexparticles. The supernatant is decanted and the latex particlesre-dispersed in an additional 10 ml of MES. This mixture is centrifugedat 3,000×g to sediment the latex particles; and, the resultantsupernatant is discarded and the pellet resuspended in 5 ml of MES toyield a latex suspension of approximately 2% solids. An equal volume ofthe 2% latex suspension is added to a 1 mg eER-LBD/ml MES solution(assuming latex particle size is 1 μM). The concentration of eER-LBD canbe scaled up or down for smaller and larger particle sizes,respectively. The latex particle/eER-LBD mixture is incubated overnightwith gentle mixing at room temperature. The unbound eER-LBD is separatedfrom the eER-LBD-labeled latex particles by centrifugation. Thesupernatant is saved and a protein determination is performed using theMicro BCA Protein Determination Kit (Pierce). The pellet is resuspendedin 10 ml of phosphate-buffered saline, pH=7.2 (PBS) and centrifuged tosediment the particles. This washing step is repeated twice for a totalof three washes. The final latex pellet is suspended in the originalcoupling volume (final concentration of 1.0% solids) of PBS amended with0.1% glycine and 0.1% sodium azide (storage buffer) and stored at 4° C.Glycine will cover any reactive sites on the microsphere surface notoccupied by eER-LBD and will ultimately reduce nonspecific binding. Theamount of eER-LBD coupled to the latex particles is determined bysubtracting the residual protein measured in the supernatant from theoriginal amount added. The binding properties of the eER-LBD-labeledlatex particles are assessed as previously described except that boundis separated from free via centrifugation.

EXAMPLE 13 Coating Latex Particles with eER-LBD by Covalent Coupling

Although passive adsorption is the preferred method of coating latexparticles, primary amino groups of protein molecules can also becovalently coupled to carboxyl functional groups on the latex particlesusing a water soluble carbodiimide such as 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide-HCl (EDAC). Latex suspensions are brought to 2%solids as described above for passive adsorption. This procedure willwork with either carboxyl latex or carboxylate-modified latex (CML). Tothe latex, 2 ml of EDAC in MES (50 mg/ml) and 3 ml of eER-LBD(approximately 5 mg of protein) is added. The latex/protein mixture isincubated at room temperature for 3–4 hours on a rocking platform.Unbound eER-LBD is removed by centrifugation and the supernatantretained for protein determination. The eER-LBD-labeled latex particlesis washed 3 times in PBS and resuspended to 1% solids in storage bufferand stored at 4° C. until used to characterize binding properties aspreviously described except that bound is separated from free viacentrifugation.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

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1. An isolated ligand binding domain of an equine estrogen receptormolecule that can bind to a target analyte specific for said ligandbinding domain, wherein said ligand binding domain consists of the aminoacid sequence of SEQ ID NO:1.